Compositions containing sertoli cells and myoid cells and use thereof in cellular transplants

This is a continuation of U.S. patent application Ser. No. 10/883,888, filed Jul. 2, 2004, which claims the benefit of U.S. Provisional Application No. 60/484,960, filed Jul. 3, 2003, which is incorporated herein by reference.

The present invention relates to the use of Sertoli cells and myoid cells for creating an immunologically privileged site in a mammalian subject, thereby facilitating the transplantation of cells that produce a biological factor in the treatment of a disease that results from a deficiency of such biological factor. Pharmaceutical compositions containing Sertoli cells and myoid cells, as well as therapeutic methods relating to the use of these cells are provided by the present invention.

The testis, brain, and anterior chamber of the eye are considered immunoprivileged sites and have been investigated for their ability to protect cellular grafts [1-3]. Allogeneic and concordant xenogeneic tissues transplanted into the testis survive long term [4-10].

Moreover, parathyroid allografts in the testis restore normocalcemia in parathyroidectomized rats [8] and islets transplanted into the intra-abdominally placed testis correct hyperglycemia in diabetic rodents [9, 10]. Immune tolerance in the testis is due at least in part to Sertoli cells; because grafts are still protected after the selective destruction of the other major components of the testis, Leydig cells and germ cells [11, 12]. Furthermore, rodent Sertoli cells can survive as allografts [13, 14] and allogeneic islets or xenogeneic adrenal chromaffin cells are protected from immune-mediated rejection when co-transplanted with Sertoli cells [14-16]. Sertoli cells comprise a major component of the mammalian testis and are considered “nurse” cells because they provide numerous factors required for the orderly development and protection of spermatozoa [17]. As the germ cells mature they develop surface antigens which are recognized as foreign by the immune system making it necessary for the testis to develop a mechanism for protecting the developing germinal cells [18, 19]. It is believed that Sertoli cells protect the germ cells by creation of the blood-testis barrier [20, 21] and secretion of factors that may lead to local immune tolerance [13, 22-26]. Sertoli cells are known to produce Fas ligand (FasL) [13], transforming growth factor b (TGF β) [27], and clusterin [28], which are believed to have immunoprotective [13, 29], anti-inflammatory [30, 31], and tolerogenic properties [28, 32], respectively. It is postulated that these proteins may be responsible for the immunoprotective ability of Sertoli cells. Further study of these factors in an established model of Sertoli cell transplantation may provide clues to factors necessary for creating a local tolerogenic environment.

Recently, it has been shown that an immunoprivileged site can be created by preengrafting Sertoli cells, which site subsequently protected allogeneic islets from rejection [56]. Long-term survival of porcine islets placed in the repositioned intra-abdominal testis was reported in non-immunosuppressed beagle dogs [57]. Survival of syngeneic rat islet grafts transplanted in the omental pouch was also reported [58]. A model for studying islet development and xenotransplantation has been described [59]. Recent reports have shown the survival of Sertoli cells from 12-week-old Yorkshire pigs in the rat brain, an immunoprivileged site [33]. However, survival of discordant xenogeneic porcine Sertoli cells has not been demonstrated in a non-immunoprivileged site.

The present inventors have demonstrated a long-term survival of neonatal porcine Sertoli cells (NPSCs) in non-immunosuppressed Lewis rats when transplanted underneath the kidney capsule, a non-immunoprivilaged site. The present inventors have surprisingly found that the long-term survival of Sertoli cells depends upon the presence of myoid cells.

Accordingly, one embodiment of the present invention provides a pharmaceutical composition containing Sertoli cells, myoid cells and a pharmaceutically acceptable carrier.

In a preferred embodiment, the ratio of myoid cells versus Sertoli cells in the pharmaceutical composition is at least about 0.5:99.5. Preferably, the ratio is in the range of 0.5:99.5 to 65:35.

Sertoli cells and myoid cells each can be isolated from the testis of a mammal, preferably a pig, and more preferably a neonatal pig of 60 days old or younger, and even more preferably, a neonatal pig of 5 days old or younger. Alternatively, Sertoli cells can be obtained from a cell line, which can be either an established cell line such as TM4, or a stem cell line that could be derived from human tissue.

The pharmaceutical composition of the present invention can also include cells that produce a biological factor. In a preferred embodiment, cells that produce a biological factor are cells of pancreatic islets. As to the relative amount of Sertoli cells versus cells of pancreatic islets in the pharmaceutical composition, it is preferred that not more than 2×10⁶ Sertoli cells, i.e., 2×10⁶ or fewer Sertoli cells, are used per 800 islets.

In another preferred embodiment, the pharmaceutical composition is provided in a device suitable for implantation into a mammalian subject.

In another embodiment, the present invention provides a method of creating an immunologically privileged site in a mammalian subject, preferably a human subject, by administering Sertoli-cells and myoid cells into the subject.

In a preferred embodiment, the ratio of myoid cells versus Sertoli cells administered to the subject is at least about 0.5:99.5. Preferably, the ratio is in the range of 0.5:99.5 to 65:35.

Sertoli cells and myoid cells used in the administration can each be isolated from the testis of a mammal, preferably, a pig, and more preferably, a neonatal pig of 60 days old or younger, and even more preferably, a neonatal pig of 5 days old or younger. Alternatively, Sertoli cells used in the administration can be obtained from a Sertoli cell line, either an established cell line such as TM4, or a stem cell line that could be derived from human tissue. Such Sertoli cells prepared from a cell line can be admixed with myoid cells for administration to a subject.

Preferably, Sertoli cells and myoid cells are co-cultured prior to administration under conditions, e.g., for a period of about 24-48 hours, to form Sertoli-myoid cell aggregates.

Sertoli-cells and myoid cells can be administered subcutaneously into a site in the subject or administered intramuscularly. Preferably, the site is selected from the brain, the renal subcapsular space, the liver subcapsular space, the hepatic portal vein, the omental pouch, or the subcutaneous fascia.

According to the present invention, the total amount of Sertoli-cells and myoid cells administered is in the range of 10⁵ to 10⁸ cells. Generally speaking, the amount of cells required to create an immunologically privileged site in a human subject is more than the amount required in a mouse, for example. For administration to a human subject, the total amount of cells administered is preferably about 10⁷ to 10⁸ cells.